This proposal aims to investigate the mechanisms by which melatonin (MET) regulates MT2 melatonin receptors and assess the physiological significance of these events. The specific aims include: 1) to investigate the mechanisms by which melatonin desensitizes the MT2 melatonin receptor 2) assess the role of endogenous melatonin in regulating MT2 melatonin receptor-mediated phase shifts of circadian rhythms in the suprachiasmatic nucleus (SCN). It is hypothesized that a potential mechanism mediating the decreases in specific 2-[125 I]-iodomelatonin binding is MLTmediated desensitization of the MT2 receptor through receptor internalization. Desensitization of CHO-MT2 receptors will be addressed using cAMP functional and receptor phosphorylation assays. Using dominant negatives of G protein receptor kinases (GRK), arrestins, and dynamin, confocal microscopic and cell surface biotinylation experiments will examine the process of CHO-MT2 receptor internalization. The physiological significance of this proposed MT2 receptor desensitization will be assessed by recording the neuronal firing of suprachiasmatic nucleus (SCN) neurons in an in vitro rat brain slice. Melatonin can phase shift the peak of neuronal firing in the SCN through activation of endogenous MT2 receptors at CT10 and CT23. Desensitization of the MT2 receptor will be determined following treatment of the SCN with MLT for 8h prior to CT23 and assessing whether MLT can still phase shift the peak of neuronal firing at CT23. Internalization as a mechanism of desensitization in the SCN will be investigated in immortalized SCN cells using GFP-tagged MT2 receptors in confocal microscopic experiments. The knowledge gained from this research will advance our understanding of the involvement of MLT and the melatonin receptors in modulating circadian rhythms, whose alteration has been noted in depressive and sleep disorders.